Methionine and homocysteine modulate the rate of ROS generation of isolated mitochondria in vitro

Short term methionine restriction increases hepatic global DNA methylation in adult but not young male C57BL/6J mice

MLL (KMT2A)-rearranged Acute Lymphoblastic Leukemias Are Addicted to S-Adenosyl Methionine (SAM): Implications for Therapy

Restriction of methionine in rodent diets increases energy expenditure, limits weight gain, improves insulin sensitivity, and reduces hepatic and circulating lipids. Dietary methionine restriction (MR) is implemented using diets formulated from elemental amino acids (AA) that reduce methionine content to restricted levels of ∼0.17%. However, translational implementation of dietary MR with elemental AA-based diets is confounded by poor palatability. To solve this problem and restrict methionine using intact proteins, casein was subjected to mild oxidation to selectively reduce methionine. Diets were then formulated using oxidized casein, adding back methionine to produce a final concentration of 0.17%. The biological efficacy of dietary MR using the oxidized casein diet was compared to the standard elemental MR diet. Thirty-two, 5-week old male C57BL/6J mice were divided into two groups of 16, with one group receiving the Elemental Control (El Con) diet and the other receiving the Casein Control (Cas Con) diet for 10 days. Thereafter, 8 mice continued on the El Con diet and 8 mice were switched to the Elemental MR (El MR) diet. In similar fashion, 8 mice continued on the Cas Con diet and 8 mice were switched to the Oxidized Casein (Ox Cas) MR diet. Over the subsequent 8 weeks, the behavioral, metabolic, and endocrine responses to the 4 diets were assessed to evaluate the relative efficacy of the 2 approaches to restricting dietary methionine. The El MR diet produced the expected reductions in body weight, fat mass accumulation, fasting insulin, and hepatic and circulating triglycerides, while increasing energy expenditure, serum FGF-21 levels, and weight-specific food consumption. The Ox Cas MR diet produced increases and decreases in each of these variables that paralleled the effects of the El MR diet. Collectively, these findings demonstrate that dietary MR can be effectively implemented using casein after selective oxidative reduction of methionine. Disclosure H. Fang: None. K.P. Stone: None. L. Forney: None. L.C. Sims: None. T.W. Gettys: None. Funding Pennington Foundation

Methionine (MET) restriction (MR) has been shown to arrest cancer growth and sensitizes tumors to chemotherapy. MR total parenteral nutrition (MR TPN) with a chemotherapy-containing amino acid solution ("AO-90") (lacking both MET and L-cysteine[CYS]) showed synergistic effects with 5-fluorouracil (5-FU) in tumor-bearing rats and in a Phase I clinical trial with gastrointestinal tract cancers compared to 5-FU in a MET-containing TPN. All gastric cancer patients underwent gastrectomy. Resected tumors in the AO-90 group showed significant reduction of cancer histologically, while almost no effect was seen in the control group. A Phase II clinical trial of dietary MR combined with cystemustine treatment for melanoma or glioma was carried out. Twenty-two patients (20 with metastatic melanoma and 2 with recurrent gloma) received a median of four cycles of the combination of a 1-day MR diet with cystemustine (60mg/m2) every 2weeks. This combination was well tolerated (toxicity and nutritional status). The median disease-free survival was 1.8months and the median survival was 4.6months, with two long-duration stabilizations. MET depletion in plasma was 40%. In another study, eight patients with a variety of metastatic solid tumors were enrolled in a Phase I clinical trial of a commercially available MR medical food. Participants remained on the experimental diet for an average of 17.3weeks. Plasma methionine levels fell from 21.6 to 9μm within 2weeks, a 58% decline. The only side effect was weight loss of approximately 0.5kg per week. A feasibility study combining dietary MR with a FOLFOX regimen in patients with metastatic colorectal cancer was carried out. The plasma MET concentration was reduced by dietary MR by 58% on the first day of the MR diet. Among the four patients evaluable for response, three experienced a partial response and one patient had disease stabilization. The results of the above-described clinical trials indicate the clinical potential of MR.

The effect of UV/visible/NIR light (380/450/530/650/808/1064 nm) on ROS generation, mitochondrial activity and viability is experimentally compared in human neuroblastoma cancer cells. The absorption of photons by mitochondrial photoacceptors in Complexes I, III and IV is in detail investigated by sequential blocking with selective pharmaceutical blockers. Complex I absorbs UV/blue light by heme P450, resulting in a very high rate (14 times) of ROS generation leading to cell death. Complex III absorbs green light, by cytochromes b, c1 and c, and possesses less ability for ROS production (seven times), so that only irradiation lower than 10 mW cm-2 causes an increase in cell viability. Complex IV is well-known as the primary photoacceptor for red/NIR light. Light of 650/808 nm at 10-100 mW cm-2 generates a physiological ROS level about 20% of a basal concentration, which enhance mitochondrial activity and cell survival, while 1064 nm light does not show any distinguished effects. Further, ROS generation induced by low-intensity red/NIR light is compared in neurons, immune and cancer cells. Red light seems to more rapidly stimulate ROS production, mitochondrial activity and cell survival than 808 nm. At the same time, different cell lines demonstrate slightly various rates of ROS generation, peculiar to their cellular physiology.

Methionine Restriction As a Novel Therapeutic Strategy for MLL (KMT2A)-Rearranged Acute Lymphoblastic Leukemia

Background and Aims The clinical usefulness of protein-restricted diets is known to slow the progression of obesity, fatty liver, aging and cancer. Protein-restricted diet in chronic kidney disease is known to alleviate the deterioration of the renal function. The purpose of this study is to confirm whether the reduction in new function can be improved when methionine restricted (MR) diet is supplied in the chronic kidney disease (CKD) animal model and to study the its renoprotection mechanism. Method We used four-week-old C57L/B6 to make a CKD model by administration 0.025% adenine chow mixed with R3 diet for four weeks for the control group. For the MR diet group, we supplied the mixture of 0.025% adenine with methionine/Choline Deficient Diet. We measured serum blood urea nitrogen (BUN), serum creatinine, gut permeability, stool short chain fatty acid (SCFA), kidney α-SMA and renal pathology. This study was approved by Korea University Institutional Animal Care and Use Committee (KOREA-2019-0047). Results The principal coordinates analysis of the microbiome showed the two groups have distinctive component features. The stool SCFA, especially butyrate was increased in the MR diet group. However, gut permeability using FITC-dextran showed no significant change. The kidney α-SMA and MT stain area showed that the MR diet could improve renal fibrosis. The kidney inflammation including neutrophil and macrophage infiltration was also improved in histology analysis. The macrophage phenotype was more favorable to M2 type. Serum BUN and creatinine were significantly attenuated in the MR diet group. Conclusion In conclusion, the MR diet has a reno-protective effect in adenine induced CKD. The MR diet increased butyrate production and decreased renal inflammation and fibrosis. These results shed light on the role of protein restriction diet for CKD management in the real world.

Purpose: Rare cells within tumors with stem cell-like properties (cancer stem stem cells or CSCs) play a critical role in treatment resistance and cancer recurrence because CSCs are resistant to chemotherapy. Hence, elimination of CSCs will require new treatment approaches to improve the long-term outcomes for patients with cancer. We have recently discovered that breast CSCs require the essential amino acid methionine for cell survival. Methionine is converted by the enzyme MAT2A to S-adenosylmethionine (SAM), a universal methyl donor that regulates gene expression by DNA and histone methylation. We hypothesize that the methionine dependence of CSCs is a unique metabolic vulnerability that we can target by dietary methionine restriction (MR) and/or inhibiting the enzyme that converts methionine to SAM (MAT2A). Experimental design and Results: We examined the effects of MR and/or MAT2A inhibition on the formation of CSC-enriched “mammospheres” and on the expression of CSC markers (CD44hi/C24low) in triple (ER/PR/HER2)-negative breast cancer (TNBC) cells. MR inhibited mammosphere formation, induced apoptosis and reduced the CD44hi/C24low CSC population. These effects of MR were partly rescued by addition of SAM, suggesting that CSCs are dependent on SAM for cell survival. MR resulted in robust induction of MAT2A mRNA and protein levels. Inhibition of MAT2A with cycloleucine, or by silencing MAT2A with specific siRNAs, reduced mammosphere formation and the CD44hi/C24low CSC population. Strikingly, the effects of cycloleucine or MAT2A silencing on cancer stem cells were dramatically potentiated by MR. Treatment of TNBC mammospheres with MR or cycloleucine suppressed the expression of transcriptional regulators of pluripotency and epithelial-to-mesenchymal transition, while the combination of MR and cycloleucine resulted in more robust reductions in the expression of these transcription factors than either intervention alone. Consistent with these effects, the combination of dietary MR and cycloleucine was more effective than either treatment alone at inhibiting primary tumor growth and lung metastases in a murine TNBC model. Conclusion: Our findings point to previously unrecognized metabolic vulnerability of CSCs, namely, their intrinsic dependence on methionine and SAM for cell survival. MR induces expression of MAT2A, thereby providing an explanation for the robust synergy between MR and MAT2A inhibition/silencing in supressing CSC survival. Moreover, cycloleucine potentiates the activity of dietary MR in a murine metastatic TNBC model, providing proof-of-principle in vivo evidence for this novel metabolic approach to eradicate CSCs and thereby improve long-term clinical outcomes in poor-prognosis TNBC. Citation Format: Elena Strekalova, Dmitry Malin, Dominik Hoelper, Peter Lewis, Vincent Cryns. Targeting methionine metabolism to eradicate cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-247.

The Methionine restriction (MR) diet has been shown to delay aging and extend lifespan in various model organisms. However, the long‐term effects of MR diet on the gut microbiome composition remain unclear. To study this, male mice were started on MR and control diet regimens at 6 months and continued until 22 months of age. MR mice have reduced body weight, fat mass percentage, and bone mineral density while having increased lean mass percentage. MR mice also have increased insulin sensitivity along with increasing indirect calorimetry markers such as energy expenditure, oxygen consumption, carbon dioxide production, and glucose oxidation. Fecal samples were collected at 1 week, 18 weeks, and 57 weeks after the diet onset for 16S rRNA amplicon sequencing to study the gut microbiome composition. Alpha and beta diversity metrics detected changes occurring due to the timepoint variable, but no changes were detected due to the diet variable. The results from LEfSe analysis surprisingly showed that more bacterial taxa changes were linked to age rather than diet. Interestingly, we found that the long‐term MR diet feeding induced smaller changes compared to short‐term feeding. Specific taxa changes due to the diet were observed at the 1 or 18‐week time points, including Ileibacterium, Odoribacter, Lachnoclostridium, Marinifilaceae, and Lactobacillaceae. Furthermore, there were consistent aging‐associated changes across both groups, with an increase in Ileibacterium and Erysipelotrichaceae with age, while Eubacterium_coprostanoligenes_group, Ruminococcaceae, Peptococcaceae, and Peptococcus decreased with age.

Effect of different levels of dietary methionine restriction on relieving oxidative stress and behavioral deficits in middle-aged mice fed low-, medium-, or high-fat diet

Methionine restriction diets: Unravelling biological mechanisms and enhancing brain health

ABSTRACTMethionine -an essential amino acid that has to be provided by nutrition- and its metabolite S-Adenosyl methionine (SAM) are indispensable for cell proliferation, stem cell maintenance and epigenetic regulation1–5, three processes that are central to embryonic development6. Previous studies using chronic dietary restriction of methyl donors prior to and during gestation indicated that methionine restriction (MR) is detrimental to the development or growth of the neocortex7,8, however, the consequences of acute MR have not been extensively studied. Here, we designed a dietary MR regime coinciding with the neurogenic phases of neocortex development in the mouse. Our results indicate that dietary MR for 5 days leads to a severe reduction in neocortex growth and neuronal production. In comparison, growth of the liver and heart was unaffected, highlighting an organ-specific response to MR which was also observed at the cellular and molecular levels. Progenitor cohort labeling revealed a time-dependent sensitivity to MR and cell cycle analyses indicated that after 5 days of MR, progenitors are stalled in the S/G2 phases. Unexpectedly, neocortex growth reduction induced after 5 days of MR is completely rescued at birth when switching the dam back to control diet for the remaining of gestation, uncovering a mechanism of catch-up growth. Using multiplexed imaging we probed metabolic and epigenetic markers following MR and during catch-up growth and show that pyruvate metabolism is rewired in progenitors. Altogether, our data uncover a transient state of quiescence in G2/S which is metabolically distinct from G0 quiescence and associated with efficient catch-up growth. More globally, our study highlights both the extreme sensitivity of the developing neocortex to acute dietary changes and its remarkable plasticity.

Introduction: Methionine restriction has been shown to have growth inhibitory effects in a number of tumor cell lines in vitro. Methionine restriction has previously been demonstrated to slow PC-3 prostate tumor cell growth in vivo, however not at dietary levels tolerable to humans. Up to 90% dietary methionine restriction has been demonstrated to be both tolerable and safe in a preliminary human clinical trial. We hypothesized that 80%-90% methionine restriction could limit prostate tumor cell growth in vitro and in vivo. Methods: We reduced methionine levels by 40%, 80%, 90% and 100% in prostate cancer cell lines in vitro, relative to standard RPMI medium. Cell proliferation and viability were measured using MTS and trypan blue assays, respectively. PC-3 cells were grafted into a xenograft nude mouse model and animals were randomized to standard chow, 80% or 90% methionine restricted diets. Plasma methionine levels were measured before and after the dietary intervention. Tumor volume was measured throughout the study and mice were sacrificed when tumors reached 1000 mm3. Results: Methionine restriction exerted a dose and time-dependent effect on PC-3 tumor cell proliferation and viability in vitro. Methionine restriction of 80% and 90% for a period of 6 days reduced PC-3 tumor cell line proliferation by 20% and 40%, respectively, as measured by MTS assay (p<0.0001). Following 6 days of 80% and 90% methionine restriction, PC-3 cell viability was reduced by 10% and 15%, respectively. We are currently awaiting results from our in vivo study which is underway. Discussion: Methionine restriction slows prostate tumor cell line growth and reduces cell line viability in vitro. Based upon these in vitro results we hypothesize that an 80% - 90% methionine restricted diet will slow tumor growth in vivo, and we await the results of this dietary intervention in mice. Methionine restriction at this clinically relevant level which is tolerable and safe to humans may have the potential to slow tumor growth in prostate cancer patients; however more preliminary studies are needed in order to establish whether 80%-90% methionine restriction actually does slow tumor growth in vivo and if so to understand the mechanisms by which this occurs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5255. doi:1538-7445.AM2012-5255

Eighty percent dietary methionine restriction (MetR) in rodents (without calorie restriction), like dietary restriction (DR), increases maximum longevity and strongly decreases mitochondrial reactive oxygen species (ROS) production and oxidative stress. Eighty percent MetR also lowers the degree of membrane fatty acid unsaturation in rat liver. Mitochondrial ROS generation and the degree of fatty acid unsaturation are the only two known factors linking oxidative stress with longevity in vertebrates. However, it is unknown whether 40% MetR, the relevant methionine restriction degree to clarify the mechanisms of action of standard (40%) DR can reproduce these effects in mitochondria from vital tissues of strong relevance for aging. Here we study the effect of 40% MetR on ROS production and oxidative stress in rat brain and kidney mitochondria. Male Wistar rats were fed during 7 weeks semipurified diets differing only in their methionine content: control or 40% MetR diets. It was found that 40% MetR decreases mitochondrial ROS production and percent free radical leak (by 62-71%) at complex I during forward (but not during reverse) electron flow in both brain and kidney mitochondria, increases the oxidative phosphorylation capacity of brain mitochondria, lowers oxidative damage to kidney mitochondrial DNA, and decreases specific markers of mitochondrial protein oxidation, lipoxidation, and glycoxidation in both tissues. Forty percent MetR also decreased the amount of respiratory complexes I, III, and IV and apoptosis-inducing factor (AIF) in brain mitochondria and complex IV in kidney mitochondria, without changing the degree of mitochondrial membrane fatty acid unsaturation. Forty percent MetR, differing from 80% MetR, did not inhibit the increase in rat body weight. These changes are very similar to the ones previously found during dietary and protein restriction in rats. We conclude that methionine is the only dietary factor responsible for the decrease in mitochondrial ROS production and oxidative stress, and likely for part of the longevity extension effect, occurring in DR.

High‐fat diets produce many pernicious metabolic consequences including the adverse effects on bone. Methionine restriction (MR) and collagen peptide have already been proved to influence the overall growth of mice. Several studies reported that MR can alters bone morphology including reduced bone mass and collagen peptide can improve bone development. This study was to evaluate the rescue effects of collagen peptide on MR mice bone in male mice. We researched the effects of feeding a 20% fat diet (control), a 20% fat and Methionine restriction diet (MR), a combined MR with 1% collagen peptide (MR/P) for 12 weeks and 23 weeks on bone development in male C57BL/6 mice. MR elevated bone total antioxidant capacity (T‐AOC) and GSH/GSSG, reduced MDA when compared with controls, while MR/P group was not different from MR group. At week 12, the femur length, femur diameter and BMC of MR mice significantly lower than control. However at week 23, the difference could not be observed between MR and control group. But the femur length, femur diameter and BMC of MR/P group were lower than MR group at week 12 and week 23. At week 23, the Distal femur cancelous BMD, BV/TV, Tb.N, Tb.Th was lower in MR/P group and Tb.sp was higher compared with MR group. MR/P group also exhibited decreased expression levels of Runx2, Col1, Bglap2 and reduced bone osteocalcin compared with MR group. These results indicated that MR diet slow down the bone growth rate, but with age the bone development is better than control group and MR/P diet did not improved bone development, but even worsen cancellous bone with MR diet.Support or Funding InformationThis research was supported by program of “Collaborative innovation center of food safety and quality control in Jiangsu Province”, the National Natural Science Foundation of China (No. 31571841), State Key Laboratory of Food Science and Technology of Jiangnan University in China (SKLF‐ZZB‐201609).